Method of treating pain with an antibody against netrin-4, unc5b or neogenin

ABSTRACT

The present invention provides a screening method for pain suppressors, which method is characterized by using netrin-4 and/or a netrin-4 receptor to select a substance capable of inhibiting downstream signaling from netrin-4. According to the screening method of the present invention, pain suppressors useful as a preventive or therapeutic medicine for pain can be identified. The present invention also provides a pharmaceutical composition for prevention or treatment of pain, which composition comprises, as an active ingredient, a substance capable of inhibiting downstream signaling from netrin-4.

TECHNICAL FIELD

The present invention relates to a screening method for painsuppressors. The present invention also relates to a pharmaceuticalcomposition for treatment or prevention of pain.

BACKGROUND ART

Pain has a great impact on the physical physiological functions andmental state of patients and reduces their QOL (Quality Of Life) due toits severity. The number of patients with chronic pain in the world isreported to exceed 20 million, and the overall market size of medicinesfor pain treatment in Japan, the U.S. and Europe is said to be about 2trillion yen. In addition, the number of patients with diseases whichmay cause pain, such as cancer, stroke, diabetes and AIDS, has beenincreasing, and under such circumstances, the establishment of anappropriate treatment strategy for pain is a very important medicalissue. Particularly, neuropathic pain is less sensitive to nonsteroidalantiinflammatory drugs and narcotic analgesics, and more effectivemedicines for neuropathic pain are expected to be increasingly desired.However, the pathogenesis of neuropathic pain is diverse and theunderlying molecular mechanism is very complicated. Therefore, themedicine for radical treatment of neuropathic pain is yet to bedeveloped (Non Patent Literature 1). Clarifying the molecular mechanismof the development and maintenance of neuropathic pain and therebyadvancing the development of breakthrough medicines for neuropathic painis one of the biggest medical issues in the 21st century.

One of the causes of neuropathic pain is thought to be plastic changesin neural circuits in the dorsal horn, which is present in the dorsum ofthe spinal cord (Non Patent Literature 2). Sensory inputs from theperiphery undergo various processing, such as amplification, attenuationand integration, in the dorsal horn of the spinal cord and are deliveredto the brain. Peripheral nerve injuries reportedly induce plasticchanges in neural network in the dorsal horn of spinal cord, for exampleabnormal axon collateral formation and enhanced synaptic transmission,leading to the development of pain (Non Patent Literature 3). Therefore,clarifying the molecular mechanism which regulates the plasticity ofneural network in the dorsal horn is expected to advance the developmentof a novel pain therapy.

Netrin-4 is one of the secretory proteins belonging to the netrinfamily. Netrin-4 has a structure very similar to that of the β chain ofextracellular-matrix laminin, and is known to have various rolesincluding those associated with neurite formation, cell migration, cellsurvival, angiogenesis, cancer cell growth, etc. (Non Patent Literature4). However, there is no research report on the role of netrins in theadult spinal cord, and it is completely unknown whether netrins areassociated with the pathogenesis of pain.

CITATION LIST Non Patent Literature Non Patent Literature 1:

Dworkin R H, O'Connor A B, Backonja M, Farrar J T, Finnerup N B, JensenT S, Kalso E A, Loeser J D, Miaskowski C, Nurmikko T J, Portenoy R K,Rice A S, Stacey B R, Treede R D, Turk D C, Wallace M S: Pharmacologicmanagement of neuropathic pain: evidence-based recommendations. Pain,2007 Dec. 5; 132 (3): 237-51.

Non Patent Literature 2:

Woolf C J, Salter M W: Neuronal plasticity; increasing the gain in pain.Science. 2000 Jun. 9; 288(5472): 1765-9.

Non Patent Literature 3:

Markman J D, Dworkin R H: Ion channel targets and treatment efficacy inneuropathic pain. Journal of Pain 2006; 7 (1): S38-S47.

Non Patent Literature 4:

Lai Wing Sun K, Correia J P, Kennedy T E: Netrins: versatileextracellular cues with diverse functions. Development. 2011 June; 138(11): 2153-69.

SUMMARY OF INVENTION Technical Problem

An object of the present invention is to find a molecule associated withthe pathogenesis of pain and to provide a screening method for painsuppressors. Another object of the present invention is to provide apharmaceutical composition for prevention or treatment of pain, whichcomposition comprises a novel active ingredient.

Solution to Problem

In order bo achieve the above-mentioned objects, the present inventionincludes the following.

(1) A screening method for pain suppressors, characterized by usingnetrin-4 and/or a netrin-4 receptor.(2) The method according to the above (1), characterized by selecting asubstance capable of inhibiting downstream signaling from netrin-4.(3) The method according to the above (2), wherein the substance capableof inhibiting downstream signaling from netrin-4 is a substance capableof inhibiting the expression of netrin-4 or a netrin-4 receptor.(4) The method according to the above (2), wherein the substance capableof inhibiting downstream signaling from netrin-4 is a substance capableof inhibiting the interaction between netrin-4 and a netrin-4 receptor,(5) The method according to the above (3), comprising the steps of:

bringing a test substance into contact with cells expressing netrin-4and/or a netrin-4 receptor;

measuring the expression level of the netrin4 and/or the netrin-4receptor in the cells; and

comparing the expression levels of the netrin-4 and/or the netrin-4receptor between the cells in contact with the test substance and cellsnot in contact therewith to select a substance capable of reducing theexpression level.

(6) The method according to the above (4), comprising the steps of:

bringing a test substance into contact with netrin-4 and a netrin-4receptor;

confirming the interaction between the netrin-4 and the netrin-4receptor; and

selecting a substance capable of inhibiting the interaction between thenetrin-4 and the netrin-4 receptor.

(7) The method according to any one of the above (1) to (6), wherein thenetrin-4 receptor is Unc5B or neogenin.(8) A pharmaceutical composition for prevention or treatment of pain,comprising, as an active ingredient, a substance capable of inhibitingdownstream signaling from netrin-4.(9) The pharmaceutical composition according to the above (8), whereinthe substance capable of inhibiting downstream signaling from netrin-4is a nucleic acid capable of inhibiting the expression of netrin-4 or anetrin-4 receptor.(10) The pharmaceutical composition according to the above (9), whereinthe nucleic acid is a siRNA composed of nucleotide sequences of SEQ IDNOS: 1 and 2, nucleotide sequences of SEQ ID NOS: 3 and 4, or nucleotidesequences of SEQ ID NOS: 5 and 6 as sense and antisense strands.(11) The pharmaceutical composition according to the above (8) , whereinthe substance capable of inhibiting downstream signaling from netrin-4is an antibody against netrin-4 or an antibody against a netrin-4receptor.(12) A method for prevention or treatment of pain, comprising the stepof administering, to a mammal, an effective amount of a substancecapable of inhibiting downstream signaling from netrin-4.(13) Use of a substance capable of inhibiting downstream signaling fromnetrin-4 for production of a pharmaceutical composition for preventionor treatment of pain.(14) A substance capable of inhibiting downstream signaling fromnetrin-4 for use in prevention or treatment of pain.

ADVANTAGEOUS EFFECTS OF INVENTION

According to the screening method of the present invention, painsuppressors useful as a preventive or therapeutic medicine for pain canbe identified. In addition, the pharmaceutical composition of thepresent invention is useful for prevention or treatment of pain.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the nociceptive response to mechanical stimuli in theneuropathic pain model produced using netrin-4 gene deficient rats. (A)shows the results for rats with a wild-type netrin-4 gene, (B) shows theresults for netrin-4 gene deficient rats, and (C) shows the comparisonof the results for both types of rats.

FIG. 2 shows the nociceptive response to thermal stimuli in theneuropathic pain model produced using netrin-4 gene deficient rats. (A)shows the results for rats with a wild-type netrin-4 gene, (B) shows theresults for netrin-4 gene deficient rats, and (C) shows thecomparison.of the results for both types of rats.

FIG. 3 shows the nociceptive response to mechanical stimuli in theinflammatory pain model produced using netrin-4 gene deficient rats. (A)shows the results for rats with a wild-type netrin-4 gene, (B) shows theresults for netrin-4 gene deficient rats, and (C) shows the comparisonof the results for both types of rats.

FIG. 4 shows the change of nociceptive response caused by thedownregulation of the expression of netrin-4 gene in the neuropathicpain model rats.

FIG. 5 shows the change of nociceptive response caused by thedownregulation of the expression of netrin-4 gene in the inflammatorypain model rats.

FIG. 6 shows the change in nociceptive response after intrathecaladministration of netrin-4 to the rats.

FIG. 7 shows the degree of the change in nociceptive response frombefore to after intrathecal administration of netrin-4 at the indicatedconcentrations or a thermally denatured netrin-4 to the rats.

FIG. 8 shows the immunostaining of Iba1, GFAP, CD3ε and CD45R in thelumbar cord tissue after intrathecal administration of netrin-4 to therats.

FIG. 9 shows the immunostaining of c-fos in the lumbar cord tissue afterintrathecal administration of netrin-4 to the rats.

FIG. 10 shows the change in nociceptive response between before andafter intrathecal adminstration of siRNAs of the candidate molecules asa netrin-4 receptor to the rats. (A) shows the results for the controlsiRNA-administered group, (B) shows the results for the DCCsiRNA-administered group, (C) shows the results for the Unc5BsiRNA-administered group, and (D) shows the results for the neogeninsiRNA-administered group.

FIG. 11 shows the change in nociceptive response after intrathecaladministration of netrin-4 to the rats which had been subjected tointrathecal administration of siRNAs of the candidate molecules as anetrin-4 receptor two days before. (A) shows the results for the controlsiRNA-administered group, (B) shows the results for the DCCsiRNA-administered group, (C) shows the results for the Unc5BsiRNA-administered group, and (D) shows the results for the neogeninsiRNA-administered group.

FIG. 12 shows the intergroup comparison of the degrees of the reductionin withdrawal threshold at 24 hours after intrathecal administration ofnetrin-4 to the rats which had been subjected to intrathecaladministration of siRNAs of the candidate molecules as a netrin-4receptor two days before.

FIG. 13 shows the change of nociceptive response caused by thedownregulation of the expression of Unc5B gene in the neuropathic painmodel rats.

FIG. 14 shows the immunostaining of SHP2 and NeuN in the rat lumbar cordtissue.

FIG. 15 shows the change in nociceptive response after intrathecaladministration of netrin-4 alone or a mixture of netrin-4 with itsinhibitor NSC87877 or PTPi4 to the rats.

FIG. 16 shows the intergroup comparison of the degrees of reduction inwithdrawal threshold at 24 hours after intrathecal administration ofnetrin-4 alone or a mixture of netrin-4 with its inhibitor NSC87877 orPTPi4 to the rats.

FIG. 17 shows the change of nociceptive response caused by thefunction-blocking of netrin-4 by an anti-netrin-4 antibody in theneuropathic pain model rats.

DESCRIPTION OF EMBODIMENTS

The present inventors produced a pain model using a netrin-4 genedeficient rat and examined pain responses in the model. As a result, thepresent inventors found that netrin-4 gene is a potential causative genein the pathogenesis of pain. Furthermore, the present inventors revealedthrough various verification studies that netrin-4 has a potentiatingeffect on nociceptive response in the spinal cord and that binding ofnetrin-4 to its receptor triggers downstream signal transduction fromnetrin-4 for the potentiation of the nociceptive response.

<Screening Method>

The present invention provides a screening method for pain suppressors.The screening method of the present invention is characterized by usingnetrin-4 and/or a netrin-4 receptor. That is, the screening method ofthe present invention involves the use of netrin-4 or a netrin-4receptor, or both or them. The netrin-4 and the netrin-4 receptor usedin the screening method of the present invention may be proteins orgenes. In the case where the netrin-4 and the netrin-4 receptor areproteins, the proteins may be full-length ones or functional fragmentsthereof.

The netrin-4 used in the screening method of the present invention maybe of any living organism and is not particularly limited. Preferred isa mammalian netrin-4. The mammal is preferably a human, a chimpanzee, amonkey, a dog, a cow, a mouse, a rat, a guinea pig or the like, and morepreferably a human. Information regarding the nucleotide and amino acidsequences of the genes encoding netrin-4 proteins of various animals canbe obtained from known databases (e.g., DDBJ/GenBank/EMBL) with therespective accession numbers shown in Table 1, for example.

TABLE 1 Nucleotide sequence Amino acid sequence Human NM_021229NP_067052 Monkey NM_001266055 NP_001252984 Mouse NM_021320 NP_067295 RatNM_001106780 NP_001100250 Guinea pig XM_003475882 XP_003475930

The netrin-4 receptor is not particularly limited as long as it is amolecule serving to mediate downstream signaling of nociceptive responseby interacting with the netrin-4. Specific examples of the netrin-4receptor include Unc5B and neogenin. The netrin-4 receptor used in thescreening method of the present invention may be of any living organismand is not particularly limited. Preferred is a mammalian netrin-4receptor. The mammal is preferably a human, a chimpanzee, a monkey, adog, a cow, a mouse, a rat, a guinea pig or the like, and morepreferably a human. Information regarding the nucleotide and amino acidsequences of the genes encoding Unc5B proteins of various animals can beobtained from known databases (e.g., DDBJ/GenBank/EMBL) with therespective accession numbers shown in Table 2, for example. Informationregaining the nucleotide and amino acid sequences of the genes encodingneogenin proteins of various animals can be obtained from knowndatabases (e.g., DDBJ/GenBank/EMBL) with the respective accessionnumbers shown in Table 3, for example.

TABLE 2 Nucleotide sequence Amino acid sequence Human NM_170744NP_734465 Monkey XM_001106162 XP_001106162 Mouse NM_029770 NP_084046 RatNM_022207 NP_071543 Guinea pig XM_003473767 XP_003473815

TABLE 3 Nucleotide sequence Amino acid sequence Human NM_002499NP_002490 Monkey NM_001261500 NP_001248429 Mouse NM_008684 NP_032710 RatXM_003750526 XP_003750574 Guinea pig XM_003462198 XP_003462246

The test substances to be screened are preferably nucleic acids,peptides, proteins, non-peptidic compounds, synthetic compounds,fermentation products, cell extracts, cell culture supernatants, plantextracts, mammalian tissue extracts and plasma, etc., but are notlimited to these examples. The test substances may be novel or knownsubstances. These test substances may be in the form of a salt. The saltis composed of the test substance with a physiologically acceptable acidor base.

It is preferable to select a substance capable of inhibiting downstreamsignaling from netrin-4 through the screening method of the presentinvention. The inhibition of downstream signaling from netrin-4 forpotentiation of nociceptive response can prevent the development of painand improve the QOL of patients with pain. The substance capable ofinhibiting downstream signaling from netrin-4 may be a substance capableof inhibiting the expression of netrin-4 and/or a netrin-4 receptor, asubstance capable of inhibiting the interaction between netrin-4 and anetrin-4 receptor, or the like.

In the case where the substance capable of inhibiting the expression ofnetrin-4 or a netrin-4 receptor is to be selected through the screeningmethod of the present invention, the screening method can comprise, forexample, the following steps:

bringing a test substance into contact with cells expressing netrin-4and/or a netrin-4 receptor;measuring the expression level of the netrin-4 and/or the netrin-4receptor in the cells; andcomparing the expression levels of the netrin-4 and/or the netrin-4receptor between the cells in contact with the test substance and cellsnot in contact therewith to select a substance capable of reducing theexpression level.

The cells expressing netrin-4 and/or a netrin-4 receptor may be cellsexpressing netrin-4 or a netrin-4 receptor, or cells expressing both ofthem. Such cells may be cells in the living body or culture cells. Theculture cells may be primary culcure cells or a cell line. Examples ofthe primary culture cells include cerebral cortex neurons and spinalneurons, and examples of the cell line include a Neuro2A cell line, aHEK293 cell line and a COS-7 cell line. All these cells can preferablybe used in the screening method of the present invention.

The method for bringing a test substance into contact with cells is notparticularly limited and may be any method that allows the contact ofthe test substance with cells. For example, in the case where culturecells are used, the test substance can be added to culture medium. Inanother example, the test substance can be brought into contact withcells in the living body by systemic administration such as oral,intravenous or intraperitoneal administration, local administration to atarget organ or tissue, etc. It is preferable to prepare a control groupnot in coruoci with the test substance in the screening method of thepresent invention.

For the measurement of the expression level of the netrin-4 or thenetrin-4 receptor, the protein or mRNA level of the netrin-4 or thenetrin-4 receptor may be used as an index. The protein level can bemeasured by a known method for protein quantification after proteinextraction from the cells is performed by a known method. Examples ofthe known method for protein quantification include western blot, EIA,ELISA, RIA and a method using a protein assay reagent. The mRNA levelcan be measured by a known method for mRNA quantification after RNAextraction from the cells is performed by a known method. Examples ofthe known method for mRNA quantification include northern blot, RT-PCR,quantitative RT-PCR and RNase protection assay.

When the protein or mRNA level of the netrin-4 or the netrin-4 receptorin the cells in contact with a test substance is reduced as comparedwith that in the control group, i.e., the cells not in contact with thetest substance, the test substance can be selected as a desiredsubstance. As the selection criterion, the degree of reduction of theprotein or mRNA level of the netrin-4 or the netrin-4 receptor by a testsubstance is not particularly limited, and for example, the desiredsubstance is a substance capable of reducing the protein or mRNA levelto preferably 50% or less, more preferably 25% or less as compared withthat in the cells not in contact with the substance.

In the case where the substance capable of inhibiting the interactionbetween netrin-4 and a netrin-4 receptor is to be selected through thescreening method of the present invention, the screening method cancomprise, for example, the following steps:

bringing a test substance into contact with netrin-4 and a netrin-4receptor;confirming the interaction between the netrin-4 and the netrin-4receptor; andselecting a substance capable of inhibiting the interaction between thenetrin-4 and the netrin-4 receptor.

The netrin-4 and the netrin-4 receptor to be used may be native orrecombinant proteins. In the case of using native proteins, the desirednative proteins can be obtained by a known method (for example, affinitycolumn method) from the culture supernatant or cell extract of cellsexpressing netrin-4 and a netrin-4 receptor. In the case of usingrecombinant proteins, the desired recombinant proteins can be obtainedby a known method from the culture supernatant or cell extract of cellstransfected with a netrin-4 expression vector or a netrin-4receptorexpression vector. The recombinant protein can be produced using geneticinformation (see Tables 1 to 3) obtainable from known databases (e.g.,DDBJ/GenBank/EMBL) and known recombinant techniques.

The method for bringing a test substance in contact with netrin-4 and anetrin-4 receptor is not particularly limited. In an exemplary method, areaction system containing netrin-4 and a netrin-4 receptor is prepared,and a test substance is added thereto. The contact time and temperatureare not particularly limited and can be selected as appropriate. It ispreferable to prepare a control group not in contact with the testsubstance in the screening method of the present invention.

The method for confirming the interaction between the netrin-4 and thenetrin-4 receptor is not particularly limited, and a known method thatallows the determination of the level of binding of the netrin-4 to thenetrin-4 receptor can be selected as appropriate. For example, ELISA,fluorescence polarization, etc. can preferably be used. In an exampleusing ELISA, either netrin-4 or a netrin-4 receptor is immobilized, theother one of the two and a test substance are added thereto for bindingreaction, and the level of binding of the netrin-4 to the netrin-4receptor is determined with the use of appropriate primary and secondaryantibodies.

The method for selecting a substance capable of inhibiting theinteraction between the netrin-4 and the netrin-4 receptor is notparticularly limited. For example, when the level of binding of thenetrin-4 to the netrin-4 receptor in the presence of a test substance isreduced as compared with that in the control group, i.e., in the absenceof the test substance, the test substance can be selected as a desiredsubstance. As the selection criterion, the degree of reduction of thelevel of binding of the netrin-4 to the netrin-4 receptor by a testsubstance is not particularly limited, and for example, the desiredsubstance is a substance capable of reducing the binding level topreferably 50% or less, more preferably 25% or less as compared withthat in the absence of the substance.

<Pharmaceutical Composition for Prevention or Treatment of Pain>

The present invention provides a pharmaceutical composition forprevention or treatment of pain, which composition comprises, as anactive ingredient, a substance capable of inhibiting downstreamsignaling from netrin-4. The active ingredient of the pharmaceuticalcomposition of the present invention is preferably a substance capableof inhibiting the expression of netrin-4 or a netrin-4 receptor, or asubstance capable of inhibiting the interaction between netrin-4 and anetrin-4 receptor. Preferably, the pharmaceutical composition of thepresent invention comprises, as an active ingredient, a substanceselected through the above-described screening method of the presentinvention.

The pharmaceutical composition of the present invention can beformulated in the usual manner into a dosage form containing, as anactive ingredient, a substance capable of inhibiting downstreamsignaling from netrin-4. For example, oral preparations include solid orliquid preparations, specifically a tablet (including a sugar-coatedtablet and a film-coated tablet), a pill, a granule, a powder, a capsule(including a soft capsule), a syrup, an emulsion, a suspension, etc.These preparations can be produced by known methods and usually containa carrier, a diluent and/or an excipient used in the field ofpharmaceutical formulation. For example, the carrier or excipient usedfor tablets includes lactose, starch, sucrose and magnesium stearate.Parenteral preparations include, for example, an injection and asuppository. Examples of the injection include an intravenous injection,a subcutaneous injection, an intradermal injection, an intramuscularinjection, an intravenous drip infusion and an intraarticular injection.These injections are prepared according to a known method, for example,by dissolving, suspending or emulsifying the substance capable ofinhibiting downstream signaling from netrin-4, or a salt thereof in asterile aqueous or oily liquid usually used for injections. As theaqueous liquid for injection, for example, physiological saline, anisotonic solution containing glucose and an auxiliary substance, or thelike can be used, optionally together with a suitable solubilizer suchas an alcohol (e.g., ethanol etc.), a polyalcohol (e.g., propyleneglycol, polyethylene glycol, etc.) and a nonionic surfactant (e.g.,polysorbate 80, HCO-50, etc.). As the oily liquid, for example, sesameoil, soybean oil or the like can be used, optionally together with asolubilizer such as benzyl benzoate and benzyl alcohol. The suppositoryused for rectal administration is prepared by mixing the substancecapable of inhibiting downstream signaling from netrin-4, or a saltthereof with a usual base for suppositories. The pharmaceuticalpreparations that can be obtained in the above manner are safe and lesstoxic, and therefore can be orally or parenterally administered to, forexample, humans and other mammals (e.g., rats, mice, rabbits, sheep,pigs, cows, cats, dogs, monkeys, etc.).

The active ingredient of the pharmaceutical composition of the presentinvention is preferably a peptide or an antibody capable of inhibitingthe interaction between the netrin-4 and the netrin-4 receptor. Thepeptide capable of inhibiting the interaction between the netrin-4 andthe netrin-4 receptor is, for example, a peptide capable of binding tothe netrin-4 or a peptide capable of binding to the netrin-4 receptor.The antibody capable of inhibiting the interaction between the netrin-4and the netrin-4 receptor is, for example, an antibody against thenetrin-4 or an antibody against the netrin-4receptor. The antibody maybe a polyclonal or monoclonal antibody. The antibody may be a completeantibody molecule or an antibody fragment capable of specificallybinding to an antigen of interest (for example, Fab, F(ab′)₂, Fab′, Fv,scFv, etc.). The antibody is preferably a human-like chimeric antibodyor a humanized antibody. In the case where the active ingredient of thepharmaceutical composition of the present invention is a peptide or anantibody, the pharmaceutical composition is preferably administered asan injection or an infusion containing the active ingredient and apharmaceutically acceptable carrier via a parenteral route, for example,intravenously, intramuscularly, intradermally, intraperitoneally,subcutaneously or locally.

The present inventors have demonstrated that pain is suppressed byadministration of an antibody against netrin-4 (anti-netrin-4 polyclonalantibody) to rats (see Example 4). In addition, those skilled in the artcan easily understand that an antibody against a netrin-4 receptor hasthe same effect as the netrin-4 antibody on the suppression of pain.Therefore, both an antibody against netrin-4 and an antibody against anetrin-4 receptor are useful as active ingredients of the pharmaceuticalcomposition of the present invention.

The peptide can be prepared by a solid phase synthesis method (e.g., theFmoc method and the Boc method) or a liquid phase synthesis methodaccording to a known ordinary peptide synthesis protocol. In addition,the peptide can be prepared from a transformant carrying an expressionvector containing a DNA encoding the peptide. Alternatively, in vitrocoupled transcription-translation system can also be used for thepreparation of the peptide. The C-terminus of the peptide may be acarboxyl group, a carboxylate, an amide or an ester. The amino group atthe N-terminus of the peptide may be protected by a protecting group(for example, C₁₋₆ acyl groups including a formyl group and a C₂₋₆alkanoyl group such as acetyl, etc.). The peptide may be in the form ofa salt, preferably a physiologically acceptable salt. The peptide maycontain a D-amino acid and/or an unnatural amino acid.

The polyclonal antibody can be obtained, for example, in the followingmanner. An antigen (a netrin-4 protein or a fragment thereof, or anetrin-4 receptor protein or a fragment thereof) is dissolved in PBS andif needed further mixed with an appropriate amount of a usual adjuvant(for example, Freund's complete adjuvant) to prepare an immunogen, and amammal (e.g., a mouse, a rat, a rabbit, a goat, a horse, etc.) isimmunized with the immunogen. The immunization method is notparticularly limited, but preferred is subcutaneous or intraperitonealinjection given once or repeated several times at appropriate intervals,for example. After the immunization, blood collection from the immunizedanimal, serum separation and purification of polyclonal antibodyfractions are performed in a usual manner to give a polyclonal antibodyof interest. The monoclonal antibody can be obtained by fusing immunecells (for example, splenocytes) obtained from the above-mentionedimmunized mammal with myeloma cells to produce a hybridoma, culturingthe hybridoma and collecting an antibody from the culture. As themonoclonal antibody, a recombinant one can also be produced byrecombinant techniques, specifically by cloning an antibody gene fromthe hybridoma, inserting the gene into a suitable vector andtransfecting the vector into host cells. The phage display method canalso be used for production of the monoclonal antibody.

The human-like chimeric antibody refers to an antibody consisting of theheavy- and light-chain variable regions from a non-human animalantibody, and the heavy- and light-chain constant regions from a humanantibody. The humanized antibody refers to an antibody preparing bygrafting the complementarity determining regions (CDRs) from a non-humananimal antibody into a human antibody, and is also called a CDR-graftedantibody, a reshaped antibody, etc. The framework regions (FRs) of thehumanized antibody are selected so that the CDRs can form a favorableparatope. If needed, an amino acid(s) in the amino acid sequences of FRsin the variable region of the humanized antibody may be substituted by adifferent one(s) so that the CBRs can form an appropriate paratope.Information regarding the amino acid sequence of the constant region ofa human antibody can be obtained from known databases (e.g., ProteinData Bank etc.).

The active ingredient of the pharmaceutical composition of the presentinvention is preferably a nucleic acid capable of inhibiting theexpression of netrin-4 or a netrin-4 receptor. The nucleic acid capableof inhibiting the expression of netrin-4 or a netrin-4 receptor is, forexample, a siRNA (short interfering RNA) of a netrin-4 gene or anetrin-4 receptor gene, a shRHA (short hairpin RNA) of a netrin-4 geneor a netrin-4 receptor gene, an antisense oligonucleotide of a netrin-4gene or a netrin-4 receptor gene, or the like. Information regarding thenucleotide sequence of the netrin-4 gene or the netrin-4 receptor geneof an animal to be subjected to administration of the nucleic acid caneasily be obtained from known databases (e.g., GenBank etc.). siRNA isgenerally a double-stranded RNA of about 20 bases (for example, about 21to 23 bases) or less in length, and after expressed in cells, caninhibit the expression of its target gene (a netrin-4 gene or a netrin-4receptor gene in the present invention). shRNA is a molecule of about 20base pairs or more in which a single-stranded RNA partially contains apalindromic base sequence and thereby folds into a short hairpinstructure having a double-stranded portion and an overhang at the3′-terminus. After introduction into cells, the shRNA is degraded into aform of about 20 bases in length (typically for example, 21, 22 or 23bases) in the cells, and can inhibit the expression of its target gene(a netrin-4 gene or a netrin-4 receptor gene in the present invention)in the same manner as siRNA does. The siRNA and the shRNA may be in anyform that can inhibit the expression of a SHP-1 gene or a SHP-2 gene.The siRNA and the shRNA can be designed by a known method based on thenucleotide sequence of the target gene. The siRNA and the shRNA can beartificially produced by chemical synthesis. Alternatively, theantisense or sense RNAs can be produced in vitro, for example, bytranscription from a template DNA using T7 RNA polymerase and T7promoter. The antisense oligonucleotide may be any oligonucleotide thatis complementary to or capable of hybridizing with a contiguous 5- to100-base sequence in the DNA sequence of a netrin-4 gene or a netrin-4receptor gene. The antisense oligonucleotide may be DNA or RNA. Theantisense oligonucleotide may be modified unless its function iscompromised by such modification. The antisense oligonucleotide can beproduced in a usual manner, and for example, can easily be produced witha commercial DNA synthesizer.

In the case where the active ingredient of the pharmaceuticalcomposition of the present invention is a nucleic acid capable ofinhibiting the expression of netrin-4 or a netrin-4 receptor, it can beadministered in the form of a non-viral or viral vector. In the case ofusing a non-viral vector, a liposome-based method for introducing anucleic acid molecule (e.g., the liposome method, the HVJ-liposomemethod, the cationic liposome method, the lipofection method, thelipofectamine method, etc.), microinjection, a gene gun method forintroducing a nucleic acid molecule together with a carrier (metalparticles), and the like can be employed. In the case where a viralvector is used for siRNA or shRNA administration to a living body, viralvectors such as recombinant adenoviruses and retroviruses can be used. ADNA expressing a siRNA or shRNA of interest is introduced into adetoxified DNA or RNA virus such as detoxified retrovirus, adenovirus,adeno-associated virus, herpesvirus, vaccinia virus, poxvirus,poliovirus, Sindbis virus, Sendai virus and SV40, and cells or tissuesare infected with the resulting recombinant virus for introduction ofthe gene of interest into the cells or tissues.

The present inventors have demonstrated that administration of siRNAs ofthe rat netrin-4 gene to rats suppresses pain (see Example 1). Inaddition, the present inventors have demonstrated that administration ofsiRNAs of the rat Unc5B or neogenin gene, which is a netrin-4 receptorgene, to rats suppresses pain (see Example 2). Therefore, a siRNA of anetrin-4 gene, a siRNA of a Unc5B gene and a siRNA of a neogenin geneare useful as active ingredients of the pharmaceutical composition ofthe present invention.

The human nucleotide sequences corresponding to the target sequences ofthe siRNAs for rat genes actually used by the present inventors in theExamples can be target sequences of siRNAs for human genes. Therefore,the nucleotide sequences at positions 1951 to 1975 and at positions 2071to 2095 of the human netrin-4 gene (SEQ ID NO: 23) can preferably beused as the target sequences of the siRNAs capable of inhibiting theexpression of the human netrin-4 gene. The nucleotide sequence atpositions 3316 to 3340 of the human neogenin gene (SEQ ID NO: 24) canpreferably be used as the target sequence of the siRNA capable ofinhibiting the expression of the human neogenin gene. The sequences ofthe siRNAs targeting the above-described nucleotide sequences are shownin Table 4. Specifically shown are a siRNA of the human netrin-4 genecomposed of the nucleotide sequences of SEQ ID NOS: 1 and 2 as sense andantisense strands; a siRNA of the human netrin-4 gene composed of thenucleotide sequences of SEQ ID NOS: 3 and 4 as sense and antisensestrands; and a siRNA of the human neogenin gene composed of thenucleotide sequences of SEQ ID NOS: 5 and 6 as sense and antisensestrands. However, the siRNA capable of inhibiting the expression ofnetrin-4 or a netrin-4 receptor is not limited to these examples. ThesiRNAs that can preferably be used as an active ingredient of thepharmaceutical composition of the present invention can be designed by aknown method based on the nucleotide sequences of the target genes(including the nucleotide sequence of the human Unc5B gene (SEQ ID NO:25)).

TABLE 4 SEQ Sense strand (5′→3′) ID Target gene Antisense strand (5′→3′)NO Human Netrin-4 UACACUCAGGUAAAUGCGAAUGUAA 1 siRNA (1)UUACAUUCGCAUUUACCUGAGUGUA 2 Human Netrin-4 AUGUUGAGGUCAAUGUGAAGAUUAA 3siRNA (2) UUAAUCUUCACAUUGACCUCAACAU 4 Human NeogeninCCCAUGUCUGAAGCUGUCCAAUUCA 5 siRNA UGAAUUGGACAGCUUCAGACAUGGG 6

The sense and antisense strands of the siRNA may be of the same ordifferent base length, and the length is 30 bases or less, preferably 25bases or less, more preferably 23 bases or less, still more preferably21 bases. The sense and antisense strands may have blunt ends or 3′overhangs. The number of bases of the overhang in each strand is 1 to 10bases, preferably 1 to 4 bases, and more preferably 1 to 2 bases. Thelengths of the two overhangs are independent from each other and may bedifferent from each other. The nucleotide(s) of the overhang may be anRNA or DNA nucleotide(s). The base(s) of the overhang is/are preferablycomplementary to that (those) of the mRNA of the target gene, but maynot be complementary thereto as long as the siRNA has the ability of RNAinterference.

The siRNA may a double-stranded RNA composed of two separate strands ora double-stranded RNA in which a single-stranded RNA has a stem-loopstructure. That is, examples of the siRNA include an RNA having a loopof 2 to 4 nucleotides at the 5′-terminus of the sense strand and at the3′-terminus of the antisense strand, and an RNA having a loop of 2 to 4nucleotides at the 3′-terminus of the sense strand and at the5′-terminus of the antisense strand. Further included is an RNA having aloop of 2 to 4 nucleotides at the 5′-terminus of the sense strand and atthe 3′-terminus of the antisense strand, and another loop of 2 to 4nucleotides at the 3′-terminus of the sense strand and at the5′-terminus of the antisense strand.

The sequence of the siRNA and the target sequence are desirably thesame, but are not necessarily the exact same as long as the siRNA caninduce RNA interference. Specifically, as long as the sequence of theantisense strand of the siRNA can hybridize with the target sequence ofthe siRNA, one to several (for example, 2, 3 or 4) mismatched base pairsbetween the two sequences may be present. That is, the siRNA may be theone which has a sequence with substitution, addition or deletion of oneto several bases compared to the target sequence and can induce RNAinterference. In addition, the siRNA may be the one which has sequenceidentity of 85% or higher, preferably 90% or higher, more preferably 95%or higher, still more preferably 98% or higher with the target sequenceand can induce RNA interference.

As long as the siRNA can induce RNA interference, the siRNA may be theone in which all the nucleotides of the sense or antisense strand aresubstituted by DNA nucleotides (hybrid form), or the one in which somenucleotides in the sense strand and/or the antisense strand aresubstituted by DNA nucleotides (chimeric form). The hybrid form ispreferably the one in which the nucleotides of the sense strand aresubstituted by DNA nucleotides. The chimeric form is, for example, theone in which some nucleotides in the downstream region (that is, the3′-terminal region of the sense strand and the 5′-terminal region of theantisense strand) are substituted by DNA nucleotides. Specific examplesof the chimeric form include the one in which both the nucleotides inthe 3′- terminal region of the sense strand and the nucleotides in the5′-terminal region of the antisense strand are substituted by DNAnucleotides, and the one in which either the nucleotides in the3′-terminal region of the sense strand or the nucleotides in the5′-terminal region of the antisense strand are substituted by DNAnucleotides. In addition, the length of the substituted nucleotides ispreferably not beyond the length corresponding to half of the RNAmolecule. For example, the length of the substituted nucleotides is 1 to13 nucleotides from the terminus, and preferably 1 to 10 nucleotidesfrom the terminus. In terms of the efficacy of RNA interference, thestability of RNA molecules, the safety and other aspects, preferred asthe chimeric form of the siRNA is, for example, the one having sense andantisense strands of 19 to 21 nucleotides in which 1 to 10, preferably 1to 8, more preferably 1 to 6 nucleotides excluding overhang nucleotidesfrom the 3′-terminus of the sense strand and 1 to 10, preferably 1 to 8,more preferably 1 to 6 nucleotides from the 5′-terminus of the antisensestrand are consecutively substituted by DNA nucleotides. In this case,it is more preferable that the number of nucleotides substituted by DNAnucleotides in the sense strand (excluding overhang nucleotides) is thesame as that in the antisense strand.

The nucleotide (ribonucleotide and deoxyribonucleotide) of the siRNA maybe a nucleotide analog having a chemically modified saccharide, baseand/or phosphate as long as the siRNA can induce RNA interference.Examples of the nucleotide analog having a modified base include5-position modified uridines or cytidines (for example,5-propynyluridine, 5-propynylcytidine, 5-methylcytidine,5-methyluridine, 5-(2-amino)propyluridine, 5-halocytidine,5-halouridine, 5-methyloxyuridine, etc.); 8-position modified adenosinesor guanosines (for example, 8-bromoguanosine etc.); deazanucleotides(for example, 7-deazaadenosine etc.); and O- or N-alkyl nucleotides (forexample, N6-methyladenosine etc.). Examples of the nucleotide analoghaving a modified saccharide include 2′-position modified nucleotideanalogs in which the 2′-OH of the ribonucleotide is substituted by H,OR, R, a halogen atom, SH, SR, NH₂, NHR, NR₂ (in which R represents analkyl, alkenyl or alkynyl group having 1 to 6 carbon atoms), CN or thelike, and 5′-phosphorylated nucleotide analogs in which the 5′-terminusis mono-phosphorylated. Examples of the nucleotide analog having amodified phosphate include the ones in which a phosphoester bond to theadjacent ribonucleotide is substituted by a phosphorothioate bond.

The pharmaceutical composition of the present invention can comprise0.001 to 50% by mass, preferably 0.01 to 10% by mass, and morepreferably 0.1 to 1% by mass of the active ingredient.

The dose of the pharmaceutical composition of the present invention isappropriately determined in consideration of the purpose, the type andseverity of disease, the age, body weight, sex and medical history ofthe patient, the kind of active ingredient, etc. In the case where thesubject is an average human weighing about 65 to 70 kg, the daily doseis preferably about 0.02 to 4000 mg, and more preferably about 0.1 to200 mg. The daily total dose may be given as a single dose or dividedinto multiple doses.

The present invention includes a method for prevention or treatment ofpain, which method comprises a step of administering an effective amountof a substance capable of inhibiting downstream signaling from netrin-4.In addition, the present invention includes use of a substance capableof inhibiting downstream signaling from netrin-4 for the production of apharmaceutical composition for prevention or treatment of pain.Furthermore, the present invention includes a substance capable ofinhibiting downstream signaling from netrin-4 for use in the preventionor treatment of pain.

EXAMPLES

Hereinafter, the present invention will be illustrated in detail byexamples, but the present invention is not limited thereto.

Example 1: Association of Netrin-4 in Nociceptive Response 1-1.Experimental Method (1) Production and Breeding of Netrin-4 TransgenicAnimal

In order to clarify how netrin-4 is associated with the pathogenesis ofneuropathic pain, the pain-related behavior of transgenic rats in whichthe netrin-4 gene had been knocked out was observed. The netrin-4transgenic rats used were the same as those reported in 2007 by Dr.Kazuhiro Kitada in Graduate School of Science, Hokkaido University(Kitada K, Ishishita S, Tosaka K, Takahashi R, Ueda M, Keng V W, HorieK, Takeda J: Transposon-tagged mutagenesis in the rat. Nat. Methods.2007 February; 4 (2): 131-3), and were provided by courtesy of Dr.Kazuhiro Kitada. In the transgenic rats, a polyA sequence inserted intothe netrin-4 locus blocks the expression of a normal netrin-4 gene.

Male and female heterozygous netrin-4 transgenic rats were interbred toobtain homozygous knockout and wild-type littermates. The littermateswere bred until 8 weeks of age. For the determination of the genotype ofeach littermate, genomic DNA was extracted from the tail and subjectedto two kinds of genotyping PCR, of which one is for the detection of awild-type allele and the other is for the detection of a netrin-4-nullallele. Only the male animals were used in the pain-related behavioralexperiment because nociceptive response of female animals is known tofluctuate with the estrous cycle. Ear tags were attached to the animalsto identify their genotypes (wild-type animal, heterozygous animal andhomozygous knockout animal).

(2) Production of Neuropathic Pain Model

A neuropathic pain model was produced by partial ligation of the sciaticnerve (Seltzer Z, Dubner R, Shir Y: A novel behavioral model ofneuropathic pain disorders produced in rats by partial sciatic nerveinjury. Pain, 1990 November; 43 (2): 205-18), and used in the experimentdescribed later. The specific procedure was as follows. Under anesthesiaby inhalation of a gas mixture of isoflurane and orygen, an 8-week-oldrat was shaved on the left hind-limb thigh and groin region, which wasthen disinfected with alcohol. The skin and muscle over the jointportion of the thigh and hip bones were incised, and the sciatic nerve,which runs along with the thigh bone, was exposed. A half or one-thirdportion of the sciatic nerve was ligated with a 4-0 nylon suture, andthe muscle and skin were then sutured. In the contralateral (right) hindlimb, the skin and muscle was incised, but the sciatic nerve was keptintact for use as a sham surgery control.

(3) Production of Inflammatory Pain Model

An inflammatory pain model was produced by injection of completeFreund's adjuvant (B. B. Newbould: Chemotherapy of arthritis induced inrats by mycobacterial adjuvant. British Journal of PharmacologyChemotherapy, 1963; 21, pp. 127-136) . To be more specific, underanesthesia by inhalation of a gas mixture of isoflurane and oxygen, an8-week-old rat was subjected to injection of 40 μL of a completeFreund's adjuvant (CFA) solution into the sole of the left hindpaw.Meanwhile, the same volume of physiological saline was injected into thesole of the contralateral (right) hindpaw, which was used as a shamsurgery control.

(4) Measurement of Pain-Related Behavior

The von Frey filament test was used to measure the response tomechanical stimuli. Before the test, a plastic case was set onto ametallic mesh, and the pain model rat was put into the plastic case andacclimated to the environment for 5 to 10 minutes until settled. Afilament (Semmes-Weinstein Von Frey Anesthesiometer, Muromachi KikaiCo., Ltd.) was applied to the center of the plantar surface of thehindpaw for 3 to 5 seconds, and the threshold for hindpaw withdrawal (g)was measured. After the measurement of the withdrawal thresholds of boththe paws was completed, the animal used was identified from the ear tagand then returned to a breeding cage.

The plantar test was used to measure the response to thermal stimuli.For the test, the plantar test apparatus manufactured by Ogo Basile(model 37370) was used. Before the test, the pain model rat was put intoa plastic Case onto a glass pane (an accessory for the apparatus) andacclimated to the environment for 5 to 10 minutes until settled. Aninfrared light source (an accessory for the apparatus) was set under theglass pane, and the center of the plantar surface of the hindpaw wasirradiated from below the glass pane with infrared ray. The latency tothe first withdrawal response from the onset of the infrared irradiationwas measured. After the measurement for both the paws was completed, theanimal used was identified from the ear tag and then returned to abreeding cage.

(5) Downregulation of Netrin-4 Gene Expression by siRNA

A siRNA capable of binding to netrin-4 mRNA was intrathecallyadministered with a gene transfer reagent to downregulate the expressionof the netrin-4 gene. The specific procedure was as follows. Underanesthesia by inhalation or a gas mixture of isoflurane and oxygen, an8-week-old male Wistar rat was shaved on the back, which was thendisinfected with alcohol. A 19 G needle (TERUMO) was inserted intobetween the 5th and 6th lumbar vertebrae, and through the needle, apolyethylene tube (BECKTON DICKINSON Intramedic Polyethylene Tubing,PE-10) filled with physiological saline was inserted into the medullarycavity. In order to confirm whether the front end of the polyethylenetube was located in the lumbar enlargement of the spinal cord, 20 μL ofa 2% xylocaine (local anesthetic) injection (AstraZeneca) wasadministered to the rat from the rear end of the tube. Afterxylocaine-induced hind-limb paralysis was confirmed, the animal wasreturned to the cage. One week after the intubation, the von Freyfilament test was conducted to confirm the absence of intubation-causedmotor dysfunction or pain. A gene transfer reagent, HVJ-E(GenomeONE-Neo, Ishihara Sangyo Kaisha), was mixed with 1 μg each of twokinds of netrin-4 siRNAs (Stealth RNAi siRNA, Invitrogen, see Table 5),and 10 μL of the mixture was infused from the rear end of the tube intothe medullary cavity. Meanwhile, the same volume of a mixture of controlsiRNA (Stealth RNAi siRNA Negative Control, Invitrogen) and HVJ-E wasadministered to each animal of the control group.

(6) Preparation of Protein or Inhibitors for Intrathecal Administration

A purified netrin-4 protein (R&D) was dissolved in physiological salineto give a 40 ng/μL netrin-4 solution. HSC87877 (Calbiochem), aninhibitor of SHPs, was dissolved at a concentration of 50 mM insterilized water and kept refrigerated until use. The solution wasdiluted 50-fold with physiological saline before use. PTPi4(bis(4-trifluoromethylsulfonamidophenyl)-1,4-diisopropylben zene,Protein Tyrosine Phosphatase Inhibitor IV, Calbiochem), an inhibitor ofSHP2, was dissolved at a concentration of 16.4 mM in DMSO (dimethylsulfoxide, Sigma-Aldrich) and kept refrigerated until use. The solutionwas diluted to a final concentration of 1 mM with physiological salinebefore use.

(7) Immunohistochemical Staining

Rat tissue fixation was performed by perfusion under deep anesthesiawith intraperitoneal pentobarbital. For the fixation, 0.1 M phosphatebuffer was perfused, followed by a 4% PFA solution (a solution of 4%paraformaldehyde (Nacalai Tesque) in 0.1 M phosphate buffer). After theperfusion-fixation, the lumbar enlargement of the spinal cord wasdissected, immersed in a 4% PFA solution and further fixed for 6 hours.The 4% PFA solution was replaced with a solution of 30% sucrose (NacalaiTesque) in 0.1 M phosphate buffer before incubation with agitation at 4°C. for two consecutive days. The lumbar cord tissue was embedded in OCTcompound (Sakura Finetek Japan Co., Ltd.), frozen and sliced to athickness of 20 μm with a cryostat, and the resulting section wasmounted onto a slide glass (Matsunsmi Glass Ind., Ltd.). The specimenwas immersed in a 5% BSA solution (a solution of 5% BSA (bovine serumalbumin, Sigma-Aldrich) in 0.1 M phosphate buffer) at room temperaturefor 2 hours. The 5% BSA solution was replaced with a 5% BSA solutioncontaining a primary antibody, and the reaction was allowed to proceedat 4° C. for two consecutive days. After washing with 0.1 M phosphatebuffer 3 times, the specimen was immersed in a 5% BSA solutioncontaining a secondary antibody, and the reaction was allowed to proceedat 4° C. overnight. After washing with 0.1 M phosphate buffer 3 times,the specimen was coverslipped with a mounting medium and then observedunder a fluorescence microscope.

The antibodies used for the immunostaining were the following: anti-Iba1antibody (1:1000, Wako), anti-GFAP antibody (1:1000, Sigma-Aldrioh),anti-CD3ε antibody (1:200, eBioscience), anti-CD45R antibody (1:200,BD), anti-c-fos antibody (1:10000, Calbiochem), anti-NeuN antibody(1:1000, Millipore), anti-SHP2 antibody (1:1000, Santa Cruz),fluorescent-labeled anti-rabbit IgG antibody (1:500, Molecular Probes),and fluorescent-labeled anti-mouse IgG antibody (1:500, MolecularProbes).

1-2 Experimental Results (1) Analysis of Pain-Related Behavior ofNetrin-4 Gene Deficient Rats (1-1) Nociceptive Response to MechanicalStimuli in Neuropathic Pain Model

Male and female heterozygous netrin-4 transgenic rats were interbred toobtain homozygous knockout and wild-type littermates. Only the malelittermates were bred until 8 weeks of age. The sciatic nerve of theleft hind limb was partially ligated under inhalation anesthesia toproduce a neuropathic pain model.

In order to examine the nociceptive response to mechanical stimuli, thevon Frey filament test was conducted at 2, 4, 7, 14, 21, 28 and 35 daysafter the nerve injury. The results are shown in FIGS. 1 (A), (B) and(C). The rats with the wild-type gene showed a gradual reduction in thewithdrawal threshold of the hindpaw ipsilateral to the injury aspreviously reported, demonstrating that allodynia (hyperalgesia) hadbeen developed (FIG. 1 (A)). However, such a reduction in the withdrawalthreshold as observed in the wild-type rats was not observed in thenetrin-4 knockout rats (FIG. 1 (B)). The comparison of the withdrawalthresholds between before and after the injury revealed that thewild-type rats showed a 60% reduction in the withdrawal threshold at 2weeks after the injury while the knockout rats showed a 10% increase andno symptoms of hyperalgesia at the same time point (FIG. 1 (C)). Thecomparison of the percentage of the change in withdrawal threshold basedon the quantitative analysis showed significant differences between thewild-type rats and the homozygous knockout rats at 2, 4, 7, 14, 21 and35 days after the injury (FIG. 1 (C)) (Tukey-Kramer test, ** P<0.01, *P<0.05).

(1-2) Nociceptive Response to Thermal Stimuli in Neuropathic Pain Model

Next, the response to thermal stimuli in the neuropathic pain modelproduced using the netrin-4 knockout rats was analyzed.

Male and female heterozygous netrin-4 transgenic rats were interbred toobtain homozygous knockout and wild-type littermates. Only the malelittermates were bred until 8 weeks of age. The sciatic nerve of theleft hind limb was partially ligated under inhalation anesthesia toproduce a neuropathic pain model. The plantar test was conducted at 7,14, 21 and 28 days after the nerve injury to measure the latency to thefirst withdrawal behavior. The results are shown in FIGS. 2 (A), (B) and(C). The wild-type rats showed a significant reduction in the latency tothe first withdrawal of the hindpaw ipsilateral to the injury versus thecontralateral latency after one week had passed since the injury (FIG. 2(A)). However, such a significant reduction in the latency as observedin the wild-type rats was not observed in the netrin-4 knockout rats(FIG. 2 (B)). The comparison of the percentage of the reduction inwithdrawal latency from the level measured before the injury showedsignificant differences between the netrin-4 knockout rats and thewild-type rats at 7 and 21 days after the injury (FIG. 2 (C))(Tukey-Kramer test, ** P<0.01, * P<0.05).

(1-3) Nociceptive Response to Mechanical Stimuli in Inflammatory PainModel

An inflammatory pain model was produced by administration of completeFreund's adjuvant (CFA) as an inflammatory substance into the sole ofthe paw. The von Frey filament test was conducted at 1, 2, 4 and 7 daysafter the CFA administration. The results are shown in FIGS. 3 (A), (B)and (C). The wild-type rats showed a gradual reduction in the withdrawalthreshold on day 1 and later after the CFA administration (FIG. 3 (A)).However, such a reduction in the withdrawal threshold as observed in thewild-type rats was not observed in the netrin-4 knockout rats (FIG. 3(B)). The comparison of the percentage of the reduction in withdrawalthreshold from the level measured before the injury showed significantdifferences between the netrin-4 knockout rats and the wild-type rats at1, 2, 4 and 7 days after the CFA administration, as was the case withthe neuropathic pain model (FIG. 3 (C)) (Tukey-Kramer test, ** P<0.01, *P<0.05).

The above results show that mechanical hyperalgesia (allodynia)manifested in neither the neuropathic pain model nor the inflammatorypain model produced using the netrin-4 knockout rats. In particular, theneuropathic pain model produced using the netrin-4 knockout rats showedno thermal hyperalgesia, either. These experimental results indicate apossibility that netrin-4 gene is a causative gene in the pathogenesisof pain.

(2) Suppression of Nociceptive Response by Downregulation of Netrin-4Gene Expression

In order to clarify the effect of the downregulation of netrin-4 geneexpression on analgesia after the onset of pain, the response tomechanical stimuli after intrathecal administration of siRNAs ofnetrin-4 was examined.

(2-1) Neuropathic Pain Model Rat

A polyethylene tube was inserted into the medullary cavity of an8-week-old male Wistar rat. One week after the intubation, the sciaticnerve of the left hind limb was partially ligated to produce aneuropathic pain model. One week after the nerve injury, the von Freyfilament test was conducted to confirm that the rat had developedhyperalgesia (day 0). Netrin-4 siRNAs were mixed with the gene transferreagent HVJ-E, and the mixture was infused from the rear end of thepolyethylene tube retained in the medullary cavity. Meanwhile, a mixtureof control siRNA and HVJ-E was administered to each animal of thecontrol group. After the administration, the rear end of the tube wasclosed and the incised skin was sutured. The von Frey filament test wasconducted at 1, 2, 3 and 4 days after the siRNA administration toexamine the change in withdrawal threshold.

The results are shown in FIG. 4. The withdrawal threshold measuredbefore the siRNA administration (day 0) was low due to the onset ofneuropathic pain, but a significant increase was observed from day 2 today 3 after the administration (Tukey-Kramer test, * P<0.05).

(2-2) Inflammatory Pain Model Rat

A polyethylene tube was inserted into the medullary cavity of an8-week-old male Wistar rat. One week after the intubation, completeFreund's adjuvant (CFA) was administered into the sole of the paw toproduce an inflammatory pain model. Seven days after the CFAadministration, the von Frey filament test was conducted to confirm thatthe rat had developed hyperalgesia in the hindpaw ipsilateral to the CFAadministration (day 0). Netrin-4 siRNAs were mixed with the genetransfer reagent HVJ-E, and the mixture was infused from the rear end ofthe polyethylene tube retained in the medullary cavity. Meanwhile, amixture of control siRNA and HVJ-E was administered to each animal ofthe control group. After the administration, the rear end of the tubewas closed and the incised skin was sutured. The von Frey filament testwas conducted at 1, 2, 3 and 4 days after the siRNA administration toexamine the change in withdrawal threshold.

The results are shown in FIG. 5. The withdrawal threshold measuredbefore the siRNA administration (day 0) was low due to the onset ofneuropathic pain, but a significant increase was observed on day 2 andlater after the administration (Tukey-Kramer test, * P<0.05).

As described above, the intrathecal administration of netrin-4 siRNAsalleviated the allodynia observed after the onset of neuropathic pain orinflammatory pain, and this fact demonstrates that netrin-4 siRNA has ananalgesic effect. In particular, even after the onset of neuropathicpain, the inhibition of netrin-4 gene expression can suppress the pain.Therefore, netrin-4 can be thought to be a target molecule for paintherapy.

(3) Potentiation of Nociceptive Response by Intrathecal Administrationof Netrin-4 (3-1) Experiment 1

In order to clarify the function of netrin-4 in the spinal cord in an invivo setting, the change in nociceptive response after intrathecaladministration of netrin-4 was examined. First, a polyethylene tube wasinserted into the medullary cavity of an 8-week-old male Wistar rat. Oneweek after the intubation, the von Frey filament test was conducted toconfirm, the absence of intubation-caused motor dysfunction. Tenmicroliters of a netrin-4 solution (40 ng/μL) was infused from the rearend of the tube retained in the medullary cavity. Meanwhile, the samevolume of physiological saline was administered to each animal of thecontrol group. The von Frey filament test was conducted at 12, 24 and 48hours after the administration to examine the change in withdrawalthreshold.

The results are shown in FIG. 6. The netrin-4-administered animalsshowed a gradual reduction in the withdrawal threshold after theadministration. The comparison of the degree of the reduction inwithdrawal threshold showed a significant reduction in thenetrin-4-administered rats versus the control group at 12, 24 and 48hours after the administration (Tukey-Kramer test, ** P<0.01, * P<0.05).

(3-2) Experiment 2

Ten microliters of a netrin-4 solution (40 ng/μL), a 10% netrin-4solution (4 ng/μL) , a 1% netrin-4 solution (0.4 ng/μL) or a thermallydenatured netrin-4 solution (prepared by heat treatment of a netrin-4solution (40 ng/μL) at 100° C. for 10 minutes) was administered in thesame manner as in Experiment 1, and the degree of the change inwithdrawal threshold from before to 24 hours after the administrationwas calculated.

The results are shown in FIG. 7. The reduction in withdrawal thresholdwas observed when the concentration of netrin-4 was diluted to 10% (4ng/μL), but no significant reduction was observed when the concentrationof netrin-4 was diluted to 1% (0.4 ng/μL). The administration of thethermally denatured netrin-4 also did not reduce the withdrawalthreshold (Dunnett's test, ** P<0.01).

The above results show that intrathecal administration of netrin-4reduces the withdrawal threshold and induces hyperalgesia. This factindicates that netrin-4 in the spinal cord serves to potentiatenociceptive response in animals. It was also revealed that suchpotentiation of nociceptive response is dependent on the concentrationof netrin-4.

(3-3) Immunohistochemical Staining 1

The possibility was examined that administration of netrin-4 mightactivate glial response and immune response in the spinal cord. To thisend, netrin-4 or physiological saline was intrathecally administered,and 48 hours later, lumbar cord tissue was fixed with a 4% PFA solutionand cryosectioned at a thickness of 20 μm for immunostaining of variousmarkers.

The results are shown in FIG. 8. Iba1 is a microglial marker, GFAP is anastrocyte marker, CD3ε is a T cell marker, and CD45R is a B cell marker.No remarkable difference in cell morphology or spinal distribution ofeach type of ceil between the groups was observed. These resultsindicate that the administration of netrin-4 neither activates glialcells or immune cells in the spinal cord, nor induces hyperalgesia.

(3-4) Immunohistochemical Staining 2

It was examined whether the neuronal excitability in the spinal cordwould be changed by administration of netrin-4. Netrin-4 orphysiological saline was intrathecally administered, and 48 hours later,lumbar cord tissue was fixed with a 4% PFA solution and cryosectioned ata thickness of 20 μm for immunostaining of a neuronal activation marker,c-fos.

The results are shown in FIG. 9. FIG. 9 clearly shows that the number ofc-fos-positive cells in the dorsal horn of the spinal cord in thenetrin-4-administered rats was increased as compared with that in thecontrol group. This fact demonstrates that neuronal excitation in thedorsal horn of the spinal cord is induced by administration of netrin-4.

The above results indicate that netrin-4 serves to enhance neuronalexcitation in the dorsal horn of the spinal cord, leading topotentiation of nociceptive response.

Example 2: Identification of Receptors Serving to Transduce the Signalfrom Netrin-4 for Potentiation of Nociceptive Response

In order to clarify which kind of receptor mediates the downstreamsignaling from netrin-4 in the spinal cord for the potentiation ofnociceptive response, it was examined whether the downregulation of thegenetic expression of candidate netrin-4 receptors would cancel theeffect of intrathecal administration of netrin-4.

2-1 Experiment 1

DCC, Unc5B and neogenin are previously known to be capable of binding tonetrin-4, and siRNAs of these candidate receptor molecules wereprepared. The siRNAs of each candidate receptor were mixed with the genetransfer reagent HVJ-E, and the mixture was infused from the rear end ofthe polyethylene tube retained in the medullary cavity. Meanwhile, amixture of control siRNA (Stealth RNAi siRNA Negative Control,Invitrogen) and HVJ-E was administered to each animal of the controlgroup. After the administration, the rear end of the tube was closed andthe incised skin was sutured. The von Frey filament test was conducted 2days after the siRNA administration, and the withdrawal threshold wascompared with that measured on the previous day of the siRNAadministration. The Unc5B siRNAs used were two kinds of siRNAs shown inTable 5. The neogenin siRNAs used were two kinds of siRNAs shown inTable 5. The DCC siRNAs used were two kinds of siRNAs shown in Table 5.

TABLE 5 SEQ Sense strand (5′→3′) ID Target gene Antisense strand (5′→3′)NO Rat Netrin-4 GACACUCAGGUAAAUGUGAAUGUAA  7 siRNA (1)UUACAUUCACAUUUACCUGAGUGUC  8 Rat Netrin-4 ACGCUGAGGUCAACGUGAAGAUUAA  9siRNA (2) UUAAUCUUCACGUUGACCUCAGCGU 10 Rat Unc5BCCGUCUUUGUGGUUCUGGCAGUUCU 11 siRNA (1) AGAACUGCCAGAACCACAAAGACGG 12Rat Unc5B UCGUAAAGAACAAGCCAGUGGAAUU 13 siRNA (2)AAUUCCACUGGCUUGUUCUUUACGA 14 Rat Neogenin CCAAGCCUUAGGAUCAGCAGGGAAA 15siRNA (1) UUUCCCUGCUGAUCCUAAGGCUUGG 16 Rat NeogeninCCCAUGUCUGAAGCUGUGCAGUUCA 17 siRNA (2) UGAACUGCACAGCUUCAGACAUGGG 18Rat DCC CCACCCUUCCCAAGACUCAUGUUAA 19 siRNA (1) UUAACAUGAGUCUUGGGAAGGGUGG20 Rat DCC GAGGCUGGAGUCGAGUUCUCAUUAU 21 siRNA (2)AUAAUGAGAACUCGACUCCAGCCUC 22

The results are shown in FIGS. 10 (A) to (D). (A) is a graph for thecontrol siRNA-administered group (control group), (B) is a graph for theDCC siRNA-administered group, (C) is a graph for the Unc5BsiRNA-administered group, and (D) is a graph for the neogeninsiRNA-administered group. The DCC SiRNA-administered group, the Unc5BsiRNA-administered group and the neogenin siRNA-administered groupshowed no significant change in withdrawal threshold as compared withthat in the control group (Tukey-Kramer test).

2-2 Experiment 2

Two days after the siRNA administration, 10 μL of a solution of apurified netrin-4 protein (R&D) in physiological saline (40 ng/μL) wasinfused from the rear end of the tube retained in the medullary cavity.Meanwhile, the same volume of physiological saiine was administered toeach animal of the control group. The von Frey filament test wasconducted at 12, 24 and 48 hours after the administration to examine thechange in withdrawal threshold.

The results are shown in FIGS. 11 (A) to (D). (A) is a graph for thecontrol siRNA-administered group (control group), (B) is a graph for theDCC siRNA-administered group, (C) is a graph for the Unc5BsiRNA-administered group, and (D) is a graph for the neogeninsiRNA-administered group. The withdrawal threshold gradually reduced inthe control group after the administration of netrin-4. A similar changewas shown in the DCC siRNA-administered group. On the other hand, thereduction of withdrawal threshold caused by the administration ofnetrin-4 was suppressed in the Unc5B siRNA-administered group and in theneogenin siRNA-administered group.

FIG. 12 shows the intergroup comparison of the degrees of the reductionin withdrawal threshold at 24 hours after the administration ofnetrin-4. No significant difference was observed between the DCCsiRNA-administered group and the control group. On the other hand, thedegrees of the reduction in withdrawal threshold were significantlycurbed in the Unc5B siRNA-administered group and in the neogeninsiRNA-administered group as compared with that in the control group(Tukey-Kramer test, ** P<0.01, * P<0.05).

The above results indicate the possibility that the binding of netrin-4to Unc5B or neogenin triggers downstream signaling from netrin-4 for thepotentiation of nociceptive response.

2-3 Experiment 3

In order to clarify whether the downregulation of the expression ofUnc5B receptor gene would suppress neuropathic pain,, the Unc5B siRNAswere administered to a model rat which had developed pain, and thepain-related behavior was examined. First, a polyethylene tube wasinserted into the medullary cavity of an 8-week-old male Wistar rat. Oneweek after the intubation, the sciatic nerve of the left hind limb ofthe male rat was partially ligated to produce a neuropathic pain model.One week after the nerve irnury, the von Frey filament test wasconducted to confirm that the rat had developed hyperalgesia. Unc5BsiRNAs were mixed with the gene transfer reagent HVJ-E, and the mixturewas infused from the rear end of the polyethylene tube retained in themedullary cavity. Meanwhile, a mixture of control siRNA (Stealth RNAisiRNA Negative Control, Invitrogen) and HVJ-E was administered to eachanimal of the control group. After the administration, the rear end ofthe tube was closed and the incised skin was sutured. The von Freyfilament test was conducted at 1, 2, 3 and 4 days after the siRNAadministration to examine the change in withdrawal threshold.

The results are shown in FIG. 13. The withdrawal threshold measuredbefore the siRNA administration (day 0) was low due to the onset ofneuropathic pain, but a significant increase was observed on day 1 andlater after the administration (Tukey-Kramer test, * P<0.05). Theseresults show that the downregulation of the expression of Unc5B geneexerts an analgesic effect on neuropathic pain as was the case with thedownregulation of netrin-4 gene expression.

Example 3: Identification of Intracellular Downstream Signaling fromNetrin-4 3-1 Experiment 1

In an attempt to clarify what kind of intracellular signal is activatedvia Unc5B or neogenin by netrin-4 to potentiate nociceptive response, afocus was put on SHP2 (Src-homology 2-containing protein tyrosinephosphatase), which is a downstream molecule of netrin-4 and an enzymefor dephosphorylation of tyrosine. Firstly, the spinal distribution ofthe expressed SHP2 was analyzed. Rat lumbar cord tissue was fixed with4% PFA and cryosectioned at a thickness of 20 μm for doubleimmunostaining of SHP2 and NeuN, which are neuronal markers.

The results are shown in FIG. 14. Cells in which SHP2 and NeuN wereco-localized were observed in the dorsal horn of the spinal cord asshown in FIG. 14. These results demonstrate that SHP2 is expressed inthe neurons of the dorsal horn of the spinal cord.

3-2 Experiment 2

In order to clarify whether the activation of SHP2 is required for theaction of netrin-4 on the potentiation of nociceptive response, it wasexamined whether intrathecal administration of a SHPs inhibitor,NSC87877, or a SHP2 inhibitor, PTPi4(bis(4-trifluoromethylsulfonamidophenyl)-1,4-diisopropylben zene,Protein Tyrosine Phosphatase Inhibitor IV), would cancel the effect ofintrathecal administration of netrin-4. NSC87877 (final concentration: 1mM) or PTPi4 (final concentration: 1 mM) was mixed with netrin-4 (finalconcentration: 40 ng/μL , and 10 uL of the mixture was infused from therear end of the polyethylene tube retained in the medullary cavity. Thevon Frey filament test was conducted at 12, 24 and 48 hours after theadministration to examine the change in withdrawal threshold.

The results are shown in FIG. 15. The reduction in withdrawal thresholdwas observed in the group of administration of netrin-4 alone, but notin the group of administration of a mixture of netrin-4 and NSC87877 orPTPi4.

FIG. 16 shows the intergroup comparison of the degrees of the reductionin withdrawal threshold at 24 hours after administration. The degree ofthe reduction in withdrawal threshold was significantly curbed in thegroup of administration of netrin-4 with NSC87877 or PTPi4 as comparedwith that in the group of administration of netrin-4 alone (Tukey-Kramertest, ** P<0.01, * P<0.05).

The above results indicate that netrin-4 potentiates nociceptiveresponse through the activation of the SHP2expressed in the neurons ofthe dorsal horn of the spinal cord.

Example 4: Suppression of Nociceptive Response by Anti-Netrin-4 Antibody

In order to clarify whether the function-blocking of netrin-4 by ananti-netrin-4 antibody would exert an analgesic effect on neuropathicpain, an anti-netrin-4 antibody (R&D, AF1132) was administered to amodel rat which had developed pain, and the pain-related behavior wasexamined. First, a polyethylene tube was inserted into the medullarycavity of an 8-week-old male Wistar rat. One week after the intubation,the sciatic nerve of the left hind limb of the male rat was partiallyligated to produce a neuropathic pain model. One week after the nerveinjury, the von Frey filament test was conducted to confirm that the rathad developed hyperalgesia. A solution of an anti-netrin-4 antibody inphysiological saline (1 μg/μL) was prepared, and 30 μL of the solutionwas infused from the rear end of the polyethylene tube retained in themedullary cavity. Meanwhile, the same volume of a rat control IgGsolution (1 μg/μL) was administered to each animal of the control group.After the administration, the rear end of the tube was closed and theincised skin was sutured. The von Frey filament test was conducted at 1,2, 3 and 4 days after the antibody administration to examine the changein the withdrawal threshold of the hindpaw ipsilateral to the injury.

The results are shown in FIG. 17. The withdrawal threshold measuredbefore the anti-netrin-4 antibody administration (day 0 after theadministration) was low due to the onset of neuropathic pain, but asignificant increase was observed from day 1 to day 4 after theadministration (Tukey-Kramer test, ** P<0.01, * P<0.05). These resultsdemonstrate that the function-blocking of netrin-4 by an anti-netrin-4antibody exerts an analgesic effect on neuropathic pain as was the casein the experiment of siRNA administration.

The present invention is not limited to particular embodiments andexamples described above, and various modifications can be made withinthe scope of the appended claims. Other embodiments provided by suitablycombining technical means disclosed in separate embodiments of thepresent invention are also within the technical scope of the presentinvention. All the academic publication's and patent literature cited inthe description are incorporated herein by reference.

1-14. (canceled)
 15. A screening method for pain suppressors,characterized by using netrin-4 and/or a netrin-4 receptor to select asubstance capable of inhibiting downstream signaling from netrin-4, thenetrin-4 receptor being Unc5B or neogenin.
 16. The method according toclaim 15, wherein the substance capable of inhibiting downstreamsignaling from netrin-4 is a substance capable of inhibiting theexpression of netrin-4 or a netrin-4 receptor.
 17. The method accordingto claim 15, wherein the substance capable of inhibiting downstreamsignaling from netrin-4 is a substance capable of inhibiting theinteraction between netrin-4 and a netrin-4 receptor.
 18. The methodaccording to claim 16, comprising the steps of: bringing a testsubstance into contact with cells expressing netrin-4 and/or a netrin-4receptor; measuring the expression level of the netrin-4 and/or thenetrin-4 receptor in the cells; and comparing the expression levels ofthe netrin-4 and/or the netrin-4 receptor between the cells in contactwith the test substance and cells not in contact therewith to select asubstance capable of reducing the expression level.
 19. The methodaccording to claim 17, comprising the steps of: bringing a testsubstance into contact with netrin-4 and a netrin-4 receptor; confirmingthe interaction between the netrin-4 and the netrin-4 receptor; andselecting a substance capable of inhibiting the interaction between thenetrin-4 and the netrin-4 receptor.
 20. A method for prevention ortreatment of pain, comprising the step of administering, to a mammal, aneffective amount of a nucleic acid capable of inhibiting the expressionof netrin-4, Unc5B or neogenin.
 21. The method according to claim 20,wherein the nucleic acid is a siRNA composed of nucleotide sequences ofSEQ ID NOS: 1 and 2, nucleotide sequences of SEQ ID NOS: 3 and 4, ornucleotide sequences of SEQ ID NOS: 5 and 6 as sense and antisensestrands.